The mechanisms by which enteropathogenic E. coli (EPEC) cause diarrhea are not fully defined despite years of investigation. Tight junctions (TJs) regulate water and ion movement in the intestine and protect the sterile compartments beneath the single layer of epithelial cells from luminal contents. EPEC alter dramatically TJ structure and function by injecting effector molecules into intestinal epithelial cells via a type III secreton system (T3SS). The long-term goal of this proposal is to gain a better understanding of how multiple EPEC effectors coordinate to disrupt TJs. The independent and coordinated activities of four T3SS effector proteins (EspF, Map, EspG, NleA) will be studied. TJs remodel constantly to maintain homeostasis and undergo restoration. Preliminary data support that EPEC effectors interfere with homeostatic and restorative processes including cell polarity, TJ protein recycling, and trafficking of newly synthesized proteins. The objective, in pursuit of this goal, is to define how specific EPEC effectors cooperate to alter host cell processes that stabilize intact TJs and block those that restore disrupted TJs. The central hypothesis is that EPEC perturbation of TJs is a 2-hit process involving dismantling of intact TJ by EspF and Map, and hindrance of TJ restoration by EspG and NleA by interfering with TJ protein recycling and the transport of newly synthesized TJ proteins. In vitro and in vivo models will be used to study infection with wildtype EPEC as well as key mutant strains. State-of- the-art microscopic techniques will be coupled with molecular, cellular, microbiological, protein biochemistry, and physiological approaches to achieve the stated goals. Definition of the events by which bacterial pathogens perturb TJs and of the processes involved in TJ maintenance and restoration will guide the development of strategies aimed at preserving and restoring these important structures under adverse circumstances. The central hypothesis will be tested by addressing the following Specific Aims: 1. Define the mechanisms underlying disruption of intact TJs by EPEC and investigate the cooperative relationship between specific effectors. Aim 1 will define how EspF and Map coordinate to impact cell polarity and TJs in order to identify a single clear target for novel therapeutic interventions. 2. Determine the effect of EPEC on TJ restoration and identify the effectors and mechanisms responsible. Aim 2 will address the effect of EspG-induced microtubule disruption on TJ protein recycling and restoration as well as inhibition of ER-Golgi trafficking by NleA on restoration of TJs dismantled by other effectors, such as EspF and Map. 3. Resolve the mechanisms of TJ disruption by EPEC in an in vivo model. Aim 3 will validate the findings of Aims 1 and 2 in an in vivo model using state-of-the-art techniques. The completion of these studies will lead not only to a better understanding of EPEC pathogenesis but also to the identification of preventative and therapeutic approaches for preserving TJs.